Human macrophage metalloproteinase

ABSTRACT

A novel 54 kDa human macrophage metalloelastase (HME) having elastolytic activity and the cDNA which encodes for this enzyme are disclosed.

This is a DIVISION of application Ser. No. 08/068,392, filed May 28,1993.

BACKGROUND OF THE INVENTION

This invention relates to a novel human macrophage metalloproteinasehaving elastolytic activity and, more particularly, to the cDNA clonerepresenting the 54 kDa proenzyme referred to herein as human macrophagemetalloelastase (HME).

Metalloproteinases comprise a family of structurally related matrixdegrading enzymes involved in normal embryonic development, growth,tissue remodeling, and tissue repair.

Macrophages participate in extracellular matrix turnover both directlyby secretion of proteinases and proteinase inhibitors and indirectly bythe elaboration of cytokines such as interleukin-1 or tumor necrosisfactor, which induces proteinase production by resident fibroblasts.Human macrophages have the capacity to produce severalmetalloproteinases including interstitial collagenase, stromelysin, andtwo type IV collagenase/gelatinases of 92 and 72 kDa, of which 92 kDaenzyme represents the predominant macrophage product. The DNA cloning ofinterstitial collagenase and the two type IV-collagenase/gelatinases of72 kDa and 92 kDa is described, e.g., in U.S. Pat. Nos. 4,772,557,4,923,818 and 4,992,537, respectively. The DNA cloning of stromelysin isdescribed in PCT Int. Appln. WO 87 07,907, published Dec. 30, 1987.

Macrophages also produce the counter-regulatory tissue inhibitors ofmetalloproteinases (TIMP-1 and TIMP-2), which are proteins that inhibitmetalloproteinases via a high affinity, noncovalent bond. TIMP-2 and itshomology to TIMP-1 are described in EP 404,750, published Dec. 27, 1990and in copending U.S. application Ser. No. 07/358,043, filed May 26,1989.

Elastin degradation and abnormal repair are pivotal events in thepathogenesis of pulmonary emphysema. Cigarette smoking, the predominantrisk factor for emphysema, is associated with a large accumulation ofmacrophages in the lungs. Cigarette smoking is associated with a 10-foldincrease in cells recovered by bronchoalveolar lavage [Hunninghake andCrystal, Am. Rev. Resp. Dis. 128, 833-838 (1990)] with macrophagescomprising over 98% of cells and PMNs less than 1% [Merchant et al.,Ibid. 146, 448-453 (1992)]. Moreover, macrophages are predominant in therespiratory bronchioles of cigarette smokers where emphysematous changesare first manifest [Niewoehner et al., N. Engl. J. Med. 291, 755-758(1974)].

Recently, two classes of macrophage-derived proteinases have beenimplicated in elastolysis: (1) Matrix metalloproteinases (MMPs) that candegrade elastin include the 92 kDa gelatinase, an abundant macrophageproduct, and the 72 kDa gelatinase, secreted by many cell types butpresent in only trace amounts in macrophages [Welgus et al., J. Clin.Invest. 86, 1496-1502 (1990); Murphy and Docherty, Am. J. Respir. CellMol. Biol. 7, 120-125 (1992); and Senior et al., J. Biol. Chem. 266,7870-7875 (1991)]. Matrilysin, another recently describedmetalloproteinase with elastolytic activity, is found in peripheralblood monocytes but not in alveolar macrophages [Busiek et al., J. Biol.Chem. 267, 9087-9092 (1992)]. (2) Cysteine proteinases produced bymacrophages that cleave elastin include cathepsin L [Reilly et al.,Biochem. J. 257, 493-498 (1989)] and cathepsin S [Shi et al., J. Biol.Chem. 267, 7258-7262 (1992)], both of which are most active in acidicenvironments (˜pH 5).

It has been reported that media conditioned by mouse peritonealmacrophages exhibited significant elastolytic activity [Werb and Gordon,J. Exp. Med. 142, 361-377 (1975)]. A 22 kDa, metal-dependent,elastolytic proteinase, termed mouse macrophage elastase (MME), wasisolated [Banda and Werb, Biochem. J. 193, 589-605 (1981)]. However,despite the efforts of many investigators, human macrophage elastaseactivity could not be documented and many doubted its existence. Thepresent inventor and colleagues recently cloned the MME cDNA anddemonstrated that MME is truly a member of the MMP family [Shapiro etal., J. Biol. Chem. 267, 4664-4671 (1992)]. Surprisingly, the molecularmass of the MME proenzyme is 53 kDa, similar to several other MMPsincluding the collagenases and stromelysins. The 22 kDa active form ofMME, previously described, results from both classic N-terminalactivation and unusual C-terminal processing. MME is less than 50%identical at the amino acid level to all known human MMPs.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention the cDNA of a novel humanmacrophage metalloproteinase that possesses elastolytic activity hasbeen cloned. The primary structure of the protein, termed humanmacrophage metalloelastase (HME), has been determined and characterized.The molecular mass of the HME proenzyme is 54 kDa with a 45 kDaN-terminal active form similar to mouse macrophage metalloelastase(MME). The cDNA sequence of ˜1410 base pairs (bp) [SEQ ID NO:1] codesfor the 470 amino acids of the full HME protein [SEQ ID NO:2] which hasabout 63% homology with the MME protein [SEQ ID NO:3]. The most closelyrelated human metalloproteinases are stromelysin-1 and interstitialcollagenase, each of which has about 49% homology to HME. The isolatedHME thus is useful in a similar manner as these and other such knownmatrix metalloproteinases that have elastolytic activity. Elastolyticactivity is demonstrated herein by conventional methodology formeasuring elastin degradation by incubation with HME at 37° C.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming theinvention, it is believed that the invention will be better understoodfrom the following detailed description of preferred embodiments takenin conjunction with the appended drawings in which:

FIG. 1A shows the amino acid sequence and FIG. 1B shows the domainstructure of the 54 kDa human macrophage metalloelastase (HME). In FIG.1A the HME sequence is compared with the mouse macrophagemetalloelastase (MME) sequence.

FIG. 2 shows the Northern blot analysis of mRNA derived from humanalveolar macrophages.

FIG. 3, in two parts FIGS. 3A and 3B, shows the bacterial expression andelastolytic activity of recombinant HME. FIG. 3A shows HME cDNA ligatedinto the pET5B vector transformed and expressed in E. coli. FIG. 3B is agraphical representation that shows partially purified rHME incubated oninsoluble ³H-elastin in which elastolytic activity was quantified (μg/24hrs) by measuring radioactivity released into the medium.

FIG. 4, in two parts FIGS. 4A and 4B, shows HME secretion, processingand elastolytic activity from human alveolar macrophages. In FIG. 4A,human alveolar macrophages were obtained by bronchoalveolar lavage froma healthy cigarette smoker. FIG. 4B is a graphical representation inwhich aliquots of HME-containing conditioned media following removal ofgelatinase and inhibitors were incubated with insoluble ³H-elastin for48 hrs. at 37° C. and solubilized products were counted in ascintillation counter to estimate degradation of elastin (μg/48 hrs/10⁶cells).

FIG. 5, in four parts FIGS. 5A, 5B, 5C and 5D, shows the cDNA sequencethat codes for the HME protein of FIG. 1.

In order to illustrate specific preferred embodiments of the inventionin greater detail, the following exemplary laboratory preparative workwas carried out. However, it will be understood that the invention isnot limited to these specific examples or the details described therein.

Standard biochemical nomenclature is used herein in which the nucleotidebases are designated as adenine (A); thymine (T); guanine (G); andcytosine (C). Corresponding nucleotides are, for example,deoxyadenosine-5′-triphosphate (dATP). As is conventional forconvenience in the structural representation of a DNA nucleotidesequence, only one strand is usually shown in which A on one strandconnotes T on its complement and G connotes C. Amino acids are showneither by three letter or one letter abbreviations as follows:

Abbreviated Designation Amino Acid A Ala Alanine C Cys Cysteine D AspAspartic Acid E Glu Glutamic Acid F Phe Phenylalanine G Gly Glycine HHis Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M MetMethionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg ArginineS Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y TyrTyrosine

EXAMPLES

A distinct orthologue to mouse metalloelastase (MME) with the capacityto degrade elastin, and which represents a novel human matrixmetalloproteinase, was produced and tested as follows:

Human macrophage metalloelastase (HME) cDNA was cloned from a humanalveolar macrophage plasmid library (provided by Harold Chapman, PeterBent Brigham Hospital, Boston, Mass.) probed with a ˜400 bp genomicfragment of HME containing exons 3 and 4 that was obtained as shown inthe detailed description of FIG. 1, hereinbelow. HME cDNA is 72%identical to MME cDNA and contains an open reading frame of 1410 bp,followed by 350 bp of 3′ untranslated sequence terminating with apolyadenylation signal and a poly A-tail (FIGS. 5A to 5D). The deducedamino acid sequence (FIG. 1A) demonstrates that HME is a distinctprotein that can be organized into the typical MMP domain structurecontaining the highly conserved cysteine switch and zinc-binding motifs(FIG. 1B). The predicted molecular mass of the HME proenzyme is 54 kDawith a 45 kDa N-terminal active form, similar to MME. HME and MME are63% identical at the amino acid level. The most closely related humanMMPs are stromelysin-1 and interstitial collagenase; each is 49%identical to HME.

Northern hybridization was performed with total cellular RNA derivedfrom human alveolar macrophages. Macrophages obtained from healthycigarette smokers by bronchoalveolar lavage were maintained in culturefor 24 hrs by conventional procedures as previously described by Shapiroet al., J. Clin. Invest. 86, 1204-1210 (1990). Using HME cDNA as theprobe, a 1.8 kb mRNA species was detected (FIG. 2), confirming that thecDNA is nearly full-length and that the gene is transcribed.Furthermore, exposure of alveolar macrophages to lipopolysaccharide(LPS) increased steady-state mRNA levels, while treatment withdexamethasone reduced levels of HME mRNA. This pattern of regulation isidentical to that of MME as described by Shapiro et al., J. Biol. Chem.267, 4664-4671 (1992). HME mRNA was not detected in fresh peripheralblood monocytes.

Recombinant HME (rHME) was expressed in E. coli to determine whether HMEcDNA encoded a proteinase with elastolytic activity. Production of rHME(both proenzyme and N-terminal active forms) in crude lysates isdemonstrated in FIG. 3A by SDS/PAGE subjected to silver stain andWestern hybridization using a peptide-derived antibody specific for HME.Lysates were incubated with insoluble ³H-elastin to quantify elastolyticactivity by conventional procedures as described by Senior et al., Am.Rev. Respir. Dis. 139, 1251-1256 (1989) and Senior et al., J. Biol.Chem. 266, 7870-7875 (1991). Crude and partially purified lysatescontaining rHME were elastolytic, whereas the control lysates were not(FIG. 3B). Elastin degradation was inhibited by molar excess TIMP-1 butnot by serine or cysteine proteinase inhibitors.

It is demonstrated herein that human alveolar macrophages express andsecrete HME, and that native HME degrades elastin. Alveolar macrophagesobtained by bronchoalveolar lavage were incubated in serum containingmedium for 48 hrs. To remove both inhibitors and other releasedmetalloelastases, conditioned media was passed over gelatin- andheparin-agarose columns. The elastolytic metalloproteinases, the 92 kDaand 72 kDa gelatinases, have a gelatin binding domain while HME doesnot. Consequently, it was found that a gelatin-agarose column bound alldetectable gelatinase, but HME bound only to heparin-agarose. Partiallypurified proHME (maintained in proenzyme form with EDTA) migrated with amolecular mass of ˜54 kDa as demonstrated by Western blot (FIG. 4A).Trypsin activation converted proHME to a truncated, mature 22 kDa form,analogous to the rapid N- and C-terminal processing exhibited by MME.Partially purified native HME degraded insoluble elastin (FIG. 4B).Elastolysis was inhibited by TIMP-1 and EDTA but not by serine orcysteine proteinase inhibitors. It is estimated that HME and the 92 kDagelatinase each account for approximately one-half of macrophage-derivedmetalloelastase activity derived from the conditioned media of thealveolar macrophage cultures.

The procedures for producing and testing the disclosed MME areconveniently shown in the following detailed description of theaccompanying figures.

FIG. 1. Amino acid sequence and domain structure of HME. A. HME deducedamino acid sequence is compared with MME. These proteins are nearlyidentical in size and are 63% identical at the amino acid level, sharinghighly related cysteine-switch and zinc-binding motifs (underlined). B.HME possesses a similar domain structure to MME, the stromelysins, andthe interstitial collagenases. There is an N-terminal proenzyme domain(domain 1), a catalytic domain that coordinates the active site zincmolecule (domain II), and a C-terminal domain with weak homology tovitronectin and hemopexin (domain III). Methods: Prior to cloning humanmacrophage elastase (HME) cDNA, MME cDNA was used to screen a humangenomic library (Clontech, Palo Alto, Calif.). A 6 kB EcoR1 fragmentcontaining exons 1-4 was subcloned and sequenced. A genomic fragmentcontaining exons 3 and 4 was radiolabeled and used as the probe toscreen a human alveolar macrophage plasmid library constructed in thepcDNA 1 phagemid (provided by Harold Chapman, Peter Bent BrighamHospital, Boston, Mass.). 300,000 colonies were screened and 4 duplicatepositives were purified. The largest contained near full-length cDNAthat was subcloned into pUC9. The missing 5′ end (˜300 bp) was generatedby reverse transcription and the polymerase chain reaction (using U937RNA as the template) using reverse primers from the HME cDNA sequenceand a forward primer just upstream from the translation initiation sitebased on genomic sequence. The amplified fragment was identical to thepredicted genomic exonic sequence. This fragment was ligated in frame tothe partial HME cDNA (using an overlapping HindIII site at bp 380). Theentire cDNA (and appropriate genomic sequence) were sequenced on bothstrands using the dideoxy chain termination method of Sanger (withSequenase 2.0, United States Biochemical, Corp., Cleveland, Ohio). Thenucleotide sequence of MME is shown above the amino acid sequence inFIGS. 5A to 5D.

FIG. 2. Northern blot analysis of mRNA derived from human alveolarmacrophages. Human alveolar macrophages were obtained by bronchoalveolarlavage from a healthy smoker by conventional procedures as described byShapiro et al. J. Clin. Invest. 86, 1204-1210 (1990). Macrophages werecultured in serum containing media for 24 hrs in the presence of noadded agents (Control, Cont.), with 10⁻⁷ dexamethasone (Dex), or with2.5 μg/ml lipopolysaccharide (LPS). Total cellular RNA was thenharvested by guanidinium/acid phenol extraction, and equal amounts ofRNA were subjected to Northern blot analysis with random primer labeledfull-length HME cDNA as the probe. It is seen that HME cDNA hybridizeswith a ˜1.8 Kb mRNA, and that steady-state mRNA levels aredown-regulated by dexamethasone and up-regulated by LPS. Inset: Theethidium bromide stain of the same gel demonstrates equal quality andcontent of the loaded RNA samples.

FIG. 3. Bacterial expression and elastolytic activity of recombinantHME. A. HME cDNA ligated into the pET5B vector was transformed andexpressed in E. coli by conventional procedure as described by Shapiroet al., J. Biol. Chem. 267, 4664-4671 (1992). Crude lysates subjected toSDS polyacrylamide gel electrophoresis and silver staining demonstratethe appearance of additional bands at 54 and 45 kDa with expression ofrHME (lane 1, pET vs lane 2, pET/rHME). Western hybridizations wereperformed on denatured cell extracts in the absence (pET alone) orpresence of recombinant HME (pET/rHME). Preimmune serum failed to reactwith E. coli proteins or rHME (lanes 3 and 4). An HME-specific antibodydid not detect control E. coli proteins (pET, lane 5), but identifiedthe rHME proenzyme (54 kDa, top arrow lane 6) and N-terminal active form(45 kDa bottom arrow lane 6), as well as probable intermediate cleavageproducts. B. Partially purified rHME was incubated on insoluble³H-elastin and elastolytic activity was quantified by measuringradioactivity released into the medium by conventional procedures aspreviously described by Shapiro et al., Ibid. and Senior et al., Am.Rev. Respir. Dis. 139, 1251-1256 (1989). Control extract (pET) had noelastolytic activity, rHME-containing extract degraded significantamounts of insoluble elastin (pET/rHME). Serine and cysteine proteinaseinhibitors (Eglin C and E-64) failed to alter elastolysis but molarexcess TIMP-1 entirely inhibited pET/rHME-mediated elastin degradation.Data are expressed as micrograms of elastin degraded +/−S.D. based oncpm of triplicate samples (1 μg of elastin is represented by 1100 cpm).Similar results were obtained in 10 separate tests. Methods: Crude E.coli lysates were used for Western blots (panel A). rHME wassolubilized, refolded, and partially purified over a heparin-agarosecolumn as described for MME, Shapiro et al., Ibid., for activity (panelB). Western hybridization was performed with the ECL detection kit(Amersham, Buckinghamshire, England) using an antibody raised in rabbitsby injection of a peptide derived from the first 12 amino acids of theactive N-terminal form of HME (antibodies used at a 1/10,000 dilution).The peptide antibody (1:500 dilution) specifically reacted with the rHMEbut did not react with 2 μg of other metalloproteinases (interstitialcollagenase, stromelysin-1, matrilysin, and 92 kDa gelatinase) asdetermined by Western blotting.

FIG. 4. HME secretion, processing, and elastolytic activity from humanalveolar macrophages. A. Human alveolar macrophages were obtained bybronchoalveolar lavage from a healthy cigarette smoker as in FIG. 2,above. Macrophages were cultured in serum-containing media for 48 hrs.HME was partially purified from conditioned media as described below,and Western hybridizations were performed (as in FIG. 3). Lane 1 and 2:Preimmune serum (1:2000 dilution) blotted against proteins obtained fromconditioned media in the presence of EDTA (Lane 1) or trypsin activated(Lane 2). Lane 3: Proteins from conditioned media in the presence of 10mM EDTA (to prevent spontaneous activation) were blotted with thepeptide antibody directed against HME (1:8,000 dilution). Lane 4:Trypsin activated HME in conditioned media blotted with the peptideantibody to HME (1:8,000 dilution). It is seen that EDTA preventsactivation resulting in detection of the proenzyme migrating at amolecular mass ˜54 kDa (top arrow) and small amounts of N-terminalactivated ˜45 kDa protein (middle arrow). Trypsin activation (10 μg at25° C., 5 min., in the absence of EDTA) converts HME to the truncatedmature 22 kDa protein (lower arrow). B. Aliquots of HME-containingconditioned media following removal of gelatinase and inhibitors wereincubated with insoluble ³H-elastin for 48 hrs at 37° C. Solubilizedproducts were counted in a liquid scintillation counter to estimatedegradation of elastin (see FIG. 3). It is seen that purification andincubation with ³H-elastin in the presence of EDTA maintains theproenzyme form and prevents elastolysis. Purification in the absence ofEDTA causes spontaneous processing of HME associated with significantelastin degradation. Activity is inhibited by TIMP-1 but not serine(EglinC) or cysteine proteinase inhibitors (E-64). This activity isbelieved to be due to HME, although one cannot exclude the possiblepresence of yet another unrecognized metalloelastase with similarpurification and activation properties. Data are expressed as μg ofelastin degraded +/−S.D. of triplicate samples. Methods. Macrophageconditioned media was subjected to gelatin-agarose affinitychromatography to remove all 92 kDa and trade 72 kDa gelatinase (nogelatinase activity was detected in gelatin-agarose flow-through bygelatin zymography, <ng sensitivity). Heparin-agarose chromatography wasperformed on the flow-through fractions; HME eluted with 0.9 to 1.0 MNaCl (similar to NME). This fraction contained the predominantimmunoreactive HME and elastolytic activity present in heparin-agarosefractions eluted with a linear salt gradient. Preparation of macrophagesfrom five different volunteers who smoke cigarettes yielded identicalresults.

The invention described herein thus comprises a unique humanmetalloelastase expressed by human alveolar macrophages.Metalloproteinases (MMPs) comprise a family of structurally relatedmatrix degrading enzymes that play a major role in tissue remodeling andrepair associated with development and inflammation [Matresian, TrendsGenet. 6, 121-125 (1990); and Woessner, FASEB J. 5, 2145-2154 (1991)].Abnormal expression of MMPs can contribute to destructive processesincluding tumor invasiveness [Mignatti and Rifkin, Cell. 47, 487-498(1986) and Khokha et al., Science 243, 947-950 (1989)], arthritis (Deanet al., J. Clin. Invest. 84, 678-685 (1989) and McCachren, ArthritisRheum. 34, 1085-1093 (1991)), and atherosclerosis [Henney et al., Proc.Natl. Acad. Sci. USA, 88, 8154-8158 (1991)]. It is believed that thisnewly described metalloelastase—HME—is a candidate gene contributing tothe pathogenesis of emphysema and other inflammatory destructivediseases.

Alveolar macrophages used for the foregoing examples were derived fromhealthy cigarette smokers in order to maximize the yield. Non-smokersmay also express this enzyme, albeit from a much smaller and potentiallyless activated macrophage pool.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

3 1410 base pairs nucleic acid double linear cDNA unknown CDS 1..1410 1ATG AAG TTT CTT CTA ATA CTG CTC CTG CAG GCC ACT GCT TCT GGA GCT 48 MetLys Phe Leu Leu Ile Leu Leu Leu Gln Ala Thr Ala Ser Gly Ala 1 5 10 15CTT CCC CTG AAC AGC TCT ACA AGC CTG GAA AAA AAT AAT GTG CTA TTT 96 LeuPro Leu Asn Ser Ser Thr Ser Leu Glu Lys Asn Asn Val Leu Phe 20 25 30 GGTGAG AGA TAC TTA GAA AAA TTT TAT GGC CTT GAG ATA AAC AAA CTT 144 Gly GluArg Tyr Leu Glu Lys Phe Tyr Gly Leu Glu Ile Asn Lys Leu 35 40 45 CCA GTGACA AAA ATG AAA TAT AGT GGA AAC TTA ATG AAG GAA AAA ATC 192 Pro Val ThrLys Met Lys Tyr Ser Gly Asn Leu Met Lys Glu Lys Ile 50 55 60 CAA GAA ATGCAG CAC TTC TTG GGT CTG AAA GTG ACC GGG CAA CTG GAC 240 Gln Glu Met GlnHis Phe Leu Gly Leu Lys Val Thr Gly Gln Leu Asp 65 70 75 80 ACA TCT ACCCTG GAG ATG ATG CAC GCA CCT CGA TGT GGA GTC CCC GAT 288 Thr Ser Thr LeuGlu Met Met His Ala Pro Arg Cys Gly Val Pro Asp 85 90 95 GTC CAT CAT TTCAGG GAA ATG CCA GGG GGG CCC GTA TGG AGG AAA CAT 336 Val His His Phe ArgGlu Met Pro Gly Gly Pro Val Trp Arg Lys His 100 105 110 TAT ATC ACC TACAGA ATC AAT AAT TAC ACA CCT GAC ATG AAC CGT GAG 384 Tyr Ile Thr Tyr ArgIle Asn Asn Tyr Thr Pro Asp Met Asn Arg Glu 115 120 125 GAT GTT GAC TACGCA ATC CGG AAA GCT TTC CAA GTA TGG AGT AAT GTT 432 Asp Val Asp Tyr AlaIle Arg Lys Ala Phe Gln Val Trp Ser Asn Val 130 135 140 ACC CCC TTG AAATTC AGC AAG ATT AAC ACA GGC ATG GCT GAC ATT TTG 480 Thr Pro Leu Lys PheSer Lys Ile Asn Thr Gly Met Ala Asp Ile Leu 145 150 155 160 GTG GTT TTTGCC CGT GGA GCT CAT GGA GAC TTC CAT GCT TTT GAT GGC 528 Val Val Phe AlaArg Gly Ala His Gly Asp Phe His Ala Phe Asp Gly 165 170 175 AAA GGT GGAATC CTA GCC CAT GCT TTT GGA CCT GGA TCT GGC ATT GGA 576 Lys Gly Gly IleLeu Ala His Ala Phe Gly Pro Gly Ser Gly Ile Gly 180 185 190 GGG GAT GCACAT TTC GAT GAG GAC GAA TTC TGG ACT ACA CAT TCA GGA 624 Gly Asp Ala HisPhe Asp Glu Asp Glu Phe Trp Thr Thr His Ser Gly 195 200 205 GGC ACA AACTTG TTC CTC ACT GCT GTT CAC GAG ATT GGC CAT TCC TTA 672 Gly Thr Asn LeuPhe Leu Thr Ala Val His Glu Ile Gly His Ser Leu 210 215 220 GGT CTT GGCCAT TCT AGT GAT CCA AAG GCT GTA ATG TTC CCC ACC TAC 720 Gly Leu Gly HisSer Ser Asp Pro Lys Ala Val Met Phe Pro Thr Tyr 225 230 235 240 AAA TATGTC GAC ATC AAC ACA TTT CGC CTC TCT GCT GAT GAC ATA CGT 768 Lys Tyr ValAsp Ile Asn Thr Phe Arg Leu Ser Ala Asp Asp Ile Arg 245 250 255 GGC ATTCAG TCC CTG TAT GGA GAC CCA AAA GAG AAC CAA CGC TTG CCA 816 Gly Ile GlnSer Leu Tyr Gly Asp Pro Lys Glu Asn Gln Arg Leu Pro 260 265 270 AAT CCTGAC AAT TCA GAA CCA GCT CTC TGT GAC CCC AAT TTG AGT TTT 864 Asn Pro AspAsn Ser Glu Pro Ala Leu Cys Asp Pro Asn Leu Ser Phe 275 280 285 GAT GCTGTC ACT ACC GTG GGA AAT AAG ATC TTT TTC TTC AAA GAC AGG 912 Asp Ala ValThr Thr Val Gly Asn Lys Ile Phe Phe Phe Lys Asp Arg 290 295 300 TTC TTCTGG CTG AAG GTT TCT GAG AGA CCA AAG ACC AGT GTT AAT TTA 960 Phe Phe TrpLeu Lys Val Ser Glu Arg Pro Lys Thr Ser Val Asn Leu 305 310 315 320 ATTTCT TCC TTA TGG CCA ACC TTG CCA TCT GGC ATT GAA GCT GCT TAT 1008 Ile SerSer Leu Trp Pro Thr Leu Pro Ser Gly Ile Glu Ala Ala Tyr 325 330 335 GAAATT GAA GCC AGA AAT CAA GTT TTT CTT TTT AAA GAT GAC AAA TAC 1056 Glu IleGlu Ala Arg Asn Gln Val Phe Leu Phe Lys Asp Asp Lys Tyr 340 345 350 TGGTTA ATT AGC AAT TTA AGA CCA GAG CCA AAT TAT CCC AAG AGC ATA 1104 Trp LeuIle Ser Asn Leu Arg Pro Glu Pro Asn Tyr Pro Lys Ser Ile 355 360 365 CATTCT TTT GGT TTT CCT AAC TTT GTG AAA AAA ATT GAT GCA GCT GTT 1152 His SerPhe Gly Phe Pro Asn Phe Val Lys Lys Ile Asp Ala Ala Val 370 375 380 TTTAAC CCA CGT TTT TAT AGG ACC TAC TTC TTT GTA GAT AAC CAG TAT 1200 Phe AsnPro Arg Phe Tyr Arg Thr Tyr Phe Phe Val Asp Asn Gln Tyr 385 390 395 400TGG AGG TAT GAT GAA AGG AGA CAG ATG ATG GAC CCT GGT TAT CCC AAA 1248 TrpArg Tyr Asp Glu Arg Arg Gln Met Met Asp Pro Gly Tyr Pro Lys 405 410 415CTG ATT ACC AAG AAC TTC CAA GGA ATC GGG CCT AAA ATT GAT GCA GTC 1296 LeuIle Thr Lys Asn Phe Gln Gly Ile Gly Pro Lys Ile Asp Ala Val 420 425 430TTC TAT TCT AAA AAC AAA TAC TAC TAT TTC TTC CAA GGA TCT AAC CAA 1344 PheTyr Ser Lys Asn Lys Tyr Tyr Tyr Phe Phe Gln Gly Ser Asn Gln 435 440 445TTT GAA TAT GAC TTC CTA CTC CAA CGT ATC ACC AAA ACA CTG AAA AGC 1392 PheGlu Tyr Asp Phe Leu Leu Gln Arg Ile Thr Lys Thr Leu Lys Ser 450 455 460AAT AGC TGG TTT GGT TGT 1410 Asn Ser Trp Phe Gly Cys 465 470 470 aminoacids amino acid linear protein unknown 2 Met Lys Phe Leu Leu Ile LeuLeu Leu Gln Ala Thr Ala Ser Gly Ala 1 5 10 15 Leu Pro Leu Asn Ser SerThr Ser Leu Glu Lys Asn Asn Val Leu Phe 20 25 30 Gly Glu Arg Tyr Leu GluLys Phe Tyr Gly Leu Glu Ile Asn Lys Leu 35 40 45 Pro Val Thr Lys Met LysTyr Ser Gly Asn Leu Met Lys Glu Lys Ile 50 55 60 Gln Glu Met Gln His PheLeu Gly Leu Lys Val Thr Gly Gln Leu Asp 65 70 75 80 Thr Ser Thr Leu GluMet Met His Ala Pro Arg Cys Gly Val Pro Asp 85 90 95 Val His His Phe ArgGlu Met Pro Gly Gly Pro Val Trp Arg Lys His 100 105 110 Tyr Ile Thr TyrArg Ile Asn Asn Tyr Thr Pro Asp Met Asn Arg Glu 115 120 125 Asp Val AspTyr Ala Ile Arg Lys Ala Phe Gln Val Trp Ser Asn Val 130 135 140 Thr ProLeu Lys Phe Ser Lys Ile Asn Thr Gly Met Ala Asp Ile Leu 145 150 155 160Val Val Phe Ala Arg Gly Ala His Gly Asp Phe His Ala Phe Asp Gly 165 170175 Lys Gly Gly Ile Leu Ala His Ala Phe Gly Pro Gly Ser Gly Ile Gly 180185 190 Gly Asp Ala His Phe Asp Glu Asp Glu Phe Trp Thr Thr His Ser Gly195 200 205 Gly Thr Asn Leu Phe Leu Thr Ala Val His Glu Ile Gly His SerLeu 210 215 220 Gly Leu Gly His Ser Ser Asp Pro Lys Ala Val Met Phe ProThr Tyr 225 230 235 240 Lys Tyr Val Asp Ile Asn Thr Phe Arg Leu Ser AlaAsp Asp Ile Arg 245 250 255 Gly Ile Gln Ser Leu Tyr Gly Asp Pro Lys GluAsn Gln Arg Leu Pro 260 265 270 Asn Pro Asp Asn Ser Glu Pro Ala Leu CysAsp Pro Asn Leu Ser Phe 275 280 285 Asp Ala Val Thr Thr Val Gly Asn LysIle Phe Phe Phe Lys Asp Arg 290 295 300 Phe Phe Trp Leu Lys Val Ser GluArg Pro Lys Thr Ser Val Asn Leu 305 310 315 320 Ile Ser Ser Leu Trp ProThr Leu Pro Ser Gly Ile Glu Ala Ala Tyr 325 330 335 Glu Ile Glu Ala ArgAsn Gln Val Phe Leu Phe Lys Asp Asp Lys Tyr 340 345 350 Trp Leu Ile SerAsn Leu Arg Pro Glu Pro Asn Tyr Pro Lys Ser Ile 355 360 365 His Ser PheGly Phe Pro Asn Phe Val Lys Lys Ile Asp Ala Ala Val 370 375 380 Phe AsnPro Arg Phe Tyr Arg Thr Tyr Phe Phe Val Asp Asn Gln Tyr 385 390 395 400Trp Arg Tyr Asp Glu Arg Arg Gln Met Met Asp Pro Gly Tyr Pro Lys 405 410415 Leu Ile Thr Lys Asn Phe Gln Gly Ile Gly Pro Lys Ile Asp Ala Val 420425 430 Phe Tyr Ser Lys Asn Lys Tyr Tyr Tyr Phe Phe Gln Gly Ser Asn Gln435 440 445 Phe Glu Tyr Asp Phe Leu Leu Gln Arg Ile Thr Lys Thr Leu LysSer 450 455 460 Asn Ser Trp Phe Gly Cys 465 470 462 amino acids aminoacid linear peptide unknown 3 Met Lys Phe Leu Met Met Ile Val Phe LeuGln Val Ser Ala Cys Gly 1 5 10 15 Ala Ala Pro Met Asn Asp Ser Glu PheAla Glu Trp Tyr Leu Ser Arg 20 25 30 Phe Tyr Asp Tyr Gly Lys Asp Arg IlePro Met Thr Lys Thr Lys Thr 35 40 45 Asn Arg Asn Phe Leu Lys Glu Lys LeuGln Glu Met Gln Gln Phe Phe 50 55 60 Gly Leu Glu Ala Thr Gly Gln Leu AspAsn Ser Thr Leu Ala Ile Met 65 70 75 80 His Ile Pro Arg Cys Gly Val ProAsp Val Gln His Leu Arg Ala Val 85 90 95 Pro Gln Arg Ser Arg Trp Met LysArg Tyr Leu Thr Tyr Arg Ile Tyr 100 105 110 Asn Tyr Thr Pro Asp Met LysArg Glu Asp Val Asp Tyr Ile Phe Gln 115 120 125 Lys Ala Phe Gln Val TrpSer Asp Val Thr Pro Leu Arg Phe Arg Lys 130 135 140 Leu His Lys Asp GluAla Asp Ile Met Ile Leu Phe Ala Phe Gly Ala 145 150 155 160 His Gly AspPhe Asn Tyr Phe Asp Gly Lys Gly Gly Thr Leu Ala His 165 170 175 Val PheTyr Pro Gly Pro Gly Ile Gln Gly Asp Ala His Phe Asp Glu 180 185 190 AlaGlu Thr Trp Thr Lys Ser Phe Gln Gly Thr Asn Leu Phe Leu Val 195 200 205Ala Val His Glu Leu Gly His Ser Leu Gly Leu Gln His Ser Asn Asn 210 215220 Pro Lys Ser Ile Met Tyr Pro Thr Tyr Arg Tyr Leu Asn Pro Ser Thr 225230 235 240 Phe Arg Leu Ser Ala Asp Asp Ile Arg Asn Ile Gln Ser Leu TyrGly 245 250 255 Ala Pro Val Lys Pro Pro Ser Leu Thr Lys Pro Ser Ser ProPro Ser 260 265 270 Thr Phe Cys His Gln Ser Leu Ser Phe Asp Ala Val ThrThr Val Gly 275 280 285 Glu Lys Ile Leu Phe Phe Lys Asp Trp Phe Phe TrpTrp Lys Leu Pro 290 295 300 Gly Ser Pro Ala Thr Asn Ile Thr Ser Ile SerSer Ile Trp Pro Ser 305 310 315 320 Ile Pro Ser Ala Ile Gln Ala Ala TyrGlu Ile Glu Ser Arg Asn Gln 325 330 335 Leu Phe Leu Phe Lys Asp Glu LysTyr Trp Leu Ile Asn Asn Leu Val 340 345 350 Pro Glu Pro His Tyr Pro ArgSer Ile Tyr Ser Leu Gly Phe Ser Ala 355 360 365 Ser Val Lys Lys Val AspAla Ala Val Phe Asp Pro Leu Arg Gln Lys 370 375 380 Val Tyr Phe Phe ValAsp Lys His Tyr Trp Arg Tyr Asp Val Arg Gln 385 390 395 400 Glu Leu MetAsp Pro Ala Tyr Pro Lys Leu Phe Ser Thr His Phe Pro 405 410 415 Gly IleLys Pro Lys Ile Asp Ala Val Leu Tyr Phe Lys Arg His Tyr 420 425 430 TyrIle Phe Gln Gly Ala Tyr Gln Leu Glu Tyr Asp Pro Leu Phe Arg 435 440 445Arg Val Thr Lys Thr Leu Lys Ser Thr Ser Trp Phe Gly Cys 450 455 460

What is claimed is:
 1. Human macrophage metalloelastase in essentiallypure form and having the 470 amino acid sequence shown in SEQ ID NO:2.